1. Field of the Invention
The present invention relates to the identification and cloning of a poly(ADP-ribose) polymerase (PARP) enzyme lacking catalytic activity and methods of modulating chromatin structure.
2. Related Art
Cells within multicellular eucaryotes as they develop build complex tissue-specific chromatin architectures to express certain genes and silence others (reviewed in Farkas et al. 2000. Gene 253:117-36). These intricately acquired chromatin domains must be preserved when chromosomal DNA is accessed for replication and repair, and when reprogramming is required it must be precisely targeted. In diverse eukaryotes, protein ADP-ribosylation plays important but imperfectly understood roles in apoptosis, gene transcription and in preserving chromatin during DNA repair (De Murcia, G., and Shall, S. 2000. From DNA damage and stress signalling to cell death. Poly (ADP-Ribosylation) Reactions. (New York: Oxford University Press; Ziegler et al. 2001. Bioessays. 23: 543-548).
Poly(ADP-ribose) polymerase-1 (PARP1) is the major nuclear source of this activity in mice. The zinc fingers of the PARP1 protein specifically recognize DNA nicks and breaks and PARP activity is strongly increased upon binding to such sites. The bound, activated protein transfers multiple ADP-ribose moieties from NAD onto local chromatin proteins such as histones, topoisomerases, polymerases and transcription factors (Poirier et al. 1982. Proc. Natl. Acad. Sci. USA. 79: 3423-3427; Menissier-de Murcia et al. 1997. Proc. Natl. Acad. Sci. USA. 94: 7303-7307). These modifications facilitate base excision repair by transiently dissociating target proteins from the chromosome to expose the lesioned area, by down regulating transcription of the affected genes, and by modulating the activity of checkpoint and stress regulatory proteins. The newly repaired region returns to a normal state after PARP downregulates its own activity by automodifying a specific domain and the chromatin proteins, freed of ADP-ribose groups by a specific glycosylase, reassemble. In contrast, if damage it too extensive, PARP is specifically inactivated by caspase cleavage as the cell commits to apoptosis (Kim et al. 2000).
A great deal of biochemical and cellular evidence supports the idea that PARP removes chromatin within damaged regions to facilitate DNA repair (de Murica, 1999). Moreover, mice mutant for Parp1, one of at least five murine genes encoding PARP-related proteins, though viable and fertile, are severely compromised in their ability to repair DNA lesions (Dantzer et al. 1998. Nucleic Acids Res. 26: 1891-1898). Mice with defective PARP1 genes develop into fertile adults, hence a developmental role for PARP1 has yet to be established (Wang et al. 1995). However, four other mouse genes encode distinct ADP-ribosyl transferases with related catalytic domains (Amé et al. 1999. J. Biol. Chem. 274: 17860-17868; Kickhoefer et al. 1999. J. Cell Biol. 146: 917-928), including a telomere-associated form known as Tankyrase (Smith et al. 2001. Science. 282: 1484-1487), so functional redundancy may have obscured such a role.
Data suggesting that PARP-mediated chromatin stripping is used in other contexts has been lacking. For example, Parp1 knockout mice in addition to their damage susceptibility display dramatic immune defects, characterized by an inability to induce genes controlled by NF-κB transcription factors (Kameoka et al. 2000. Biochem J: 346:641-649). However, this defective immune response may be explained by the disruption of specific complexes that PARP forms with transcription factors such as YY1 (Oei et al. 1997. Biochem. Biophys. Res. Commun. 240:108-111), p53 (Mendoza-Alvarez et al. 2001), PAX6 (Plaza et al. 1999. Oncogene. 18:1041-1051), and NF-κB itself (Hassa et al. 1999. Biol. Chem. 380, 953-959) rather than by action at the chromatin level. Consequently, roles for PARP beyond its duties as a stress response regulator and transcriptional cofactor remain to be established.
The model eukaryote, Drosophila melanogaster, has the potential to support detailed genetic studies of PARP function in both physiology and development. Its genome contains a single gene, Parp, related to mammalian Parp1 (Uchida et al. 1993. Proc. Natl. Acad. Sci. USA. 90: 3481-3485; Hanai et al. 1998. J. Biol. Chem. 273: 11881-11886), and one homologue of tankyrase (Adams et al. 2000. Science 287: 2185-2195). The protein specified by the major Parp transcript, PARP-I, includes all the conserved domains characteristic of mammalian PARP1 except a canonical caspase cleavage site. Parp-I transcripts are expressed in nearly mature ovarian follicles and throughout embryonic development, but were not detected in larvae (Hanai et al. 1998). Parp-II transcripts lacking the automodification domain are produced via differential splicing of a single exon (Kawamura et al. 1998. Biochem Biophys Res. Commun. 251: 35-40). However, genetic studies have been hindered because Parp is located deep within centromeric heterochromatin, and its exons are scattered among several contigs that remain unlinked to the euchromatic genome sequence (Adams et al. 2000).
Drosophila development has been extensively studied to determine how changes in chromatin structure contribute to specifying programs of tissue-specific and temporally regulated gene expression (reviewed in Farkas et al. 2000; Gerasimova et al. 2001. Annu. Rev. Genet. 35: 193-208). Zygotic transcription begins during the first 14 embryonic nuclear cycles concomitant with the establishment of heterochromatin and of nucleolus formation (Foe et al. B. 1983. J. Cell Sci. 61: 51-70). During subsequent embryonic and larval stages, chromatin domains are refined under the control of multi-protein remodeling complexes (reviewed by Cairns, B. R. 1998. Trends Biochem. Sci. 23: 20-25; Jacobs et al. 1999. Semin. Cell Dev. Biol. 10: 227-235). The role of NAD-requiring enzymes in these processes is poorly known, but in addition, Parp Drosophila contain a gene structurally and functionally related to the NAD-dependent histone deacetylase encoded by the yeast Sir2 locus (Barlow et al. 2001. Exp. Cell. Res. 265: 90-103; Rosenberg et al. 2002. Cell. 109: 447-458).
However, recent genetic studies in Drosophila melanogaster show that PARP plays a much more general role by organizing chromatin at multiple points throughout the life cycle (Tulin et al. 2002). Flies bearing mutations in the single Drosophila PARP gene display extensive changes in both the repression and activation of chromosome domains, and die during the transition between the 2nd and 3rd larval instar. Heterochromatin remains abnormally accessible to nuclease, and the transcription of certain repeated sequences such as the copia retrotransposon fails to be repressed. Nucleoli are defective, and at least some specific genes seem also to misfunction as Parp mutant larvae frequently arrest development during metamorphosis. Tulin et al. (2002) proposed that the genetic requirement for PARP resulted from its involvement in locally stripping and re-assembling chromatin under developmental control. However, it is difficult to rule out that these effects were secondary to disruption of transcriptional co-activation.
Drosophila chromatin normally undergoes many highly programmed changes during embryogenesis that could be targets of PARP action (reviewed in Farkas et al. 2000). These events continue during larval development through the action of chromatin remodeling complexes (reviewed in Simon et al. 2002. Curr Opin Genet Dev. 12: 210) and histone modifications (reviewed in Wolffe and Guschin, 2000). The larval polytene chromosomes reveal that dramatic programmed chromatin alterations continue within specific euchromatic regions that form puffs at the site of newly activated genes (reviewed by Ashburner and Berendes, 1978). Many developmental puffs are induced by the moulting hormone ecdysone and contain steroid hormone response genes or their targets (reviewed by Thummel. 2000. Insect Biochem. Mol. Biol. 32:113-120).
Others puffs are rapidly induced at the sites of stress response genes following heat shock (reviewed by Farkas et al. 2000). Despite their association with induced transcription, puffs are neither necessary nor sufficient for high level gene transcription (Meyerowitz et al. 1985), and their biological significance has remained unclear.
Thus, the effects of proteins on chromatin structure are varied and influence gene transcription and expression. There is a clear need, therefore, for identification and characterization of proteins which moduldate chromatin structure, both normally and in disease states. In particular, there is a need to specifically, and in a controlled manner, manipulate chromatin structure and re-programming so as to effect expression of a gene or genes of interest in order to treat or prevent disease and/or to manipulate biological processes in vivo and in vitro.